CN101531703B - Beta lamellar blocking peptide for preventing and/or curing Alzheimer's disease - Google Patents

Abstract

The present invention relates to a class of β-sheet blocking peptides that can be used for preventing and/or treating Alzheimer's disease, and the use of the polypeptide for preventing and/or treating Alzheimer's disease.

Description

Beta-sheet blocking peptides for preventing and/or treating Alzheimer's disease

technical field

The present invention relates to polypeptides and uses thereof, more specifically, the present invention relates to a class of beta-sheet blocking peptides that can be used to prevent and/or treat Alzheimer's disease, and the polypeptides are used to prevent and/or treat Alzheimer's disease Uses for Alzheimer's disease.

Background technique

Alzheimer's disease (AD), also known as senile dementia, is a neurodegenerative disease with insidious onset and progressive course. The typical clinical features are comprehensive cognitive impairment and personality changes, manifested as Early short-term memory impairment, followed by persistent mental decline, aphasia, loss of judgment and reasoning ability, and movement disorders. This disease seriously affects the quality of life of patients and their family members, and also imposes a heavy burden on patients' families and society.

With the acceleration of population aging process, senile diseases have become a prominent problem that obviously affects human health. Senile dementia, malignant tumors, and cardiovascular and cerebrovascular accidents are listed as the three major diseases that cause the death of the elderly, and are hailed as the fourth enemy of human health in the 21st century. The World Health Organization has listed AD as one of the five key diseases in the 21st century. my country entered the aging society in 1999. At the end of 2004, my country's elderly population aged 60 and above reached 143 million, accounting for 10.97% of the total population. It is predicted that this number will reach 200 million in 2014, 300 million in 2026, exceed 400 million in 2037, reach the maximum in 2051, and then maintain the scale of 300 million to 400 million. The increasing number of elderly people makes the incidence of Alzheimer's disease relatively increase. It is understood that at present, more than 20% of the population over the age of 80 in Europe, Japan and the United States suffer from this disease. Worldwide, more than 50 million people over the age of 65 live with some form of dementia.

The main characteristics of the pathological changes of Alzheimer's disease are: a large number of senile plaques (senile plaques, SP) formed by the deposition of β-amyloid peptide (abbreviated as Aβ or βA) are formed between nerve cells, Hyperphosphorylated Tau protein causes neurofibrillary tangles (neurofibrillary tangle, NT) and massive loss of neurons. Numerous evidences indicate that neurotoxicity of Aβ is a common intersection of all etiologies. Therefore, the prevention and/or treatment targeting Aβ has become one of the focuses of AD research in recent years.

Aβ is a metabolite of β-amyloid precursor protein (APP). Under normal circumstances, APP generates soluble sAPPα under the action of α-secretase. sAPPα has the functions of reducing intracellular calcium concentration, regulating synaptic plasticity, promoting synaptic growth and protecting neurons. This is the main form of APP processing. One way does not produce Aβ; the other way is that APP produces sAPPβ and C99 under the action of β-secretase, and C99 releases Aβ peptide under the action of γ-secretase. The hydrolysis of C99 by γ-secretase is heterogeneous, and the site between alanine 713 and threonine 714 produces Aβ _1-42 ; the site between valine 711 and isoleucine 712 produces Aβ _1-40 (Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Pev, 2001, 81(2): 741-766). Aβ _1-42 accounts for about 10% of the total Aβ protein, and Aβ _1-40 accounts for about 90%, but Aβ _1-42 is more likely to aggregate, and aggregated Aβ _1-42 is the basic component of senile plaques.

The primary structure of the Aβ _1-42 peptide is shown below (SEQ ID NO: 4):

Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-

1 5 10 15

Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-Val-Gly-Ser-Asn-Lys-Gly-Ala-

16 20 25 30

Ile-Ile-Gly-Leu-Met-Val-Gty-Gly-Val-Val-Ile-Ala

31 35 40 42

The 10 amino acid residues 33-42 and 17-21 of the 10 amino acid residues at the C-terminal of Aβ _1-42 are highly hydrophobic, which constitute the hydrophobic region of Aβ _1-42 ; the 28-42 amino acid residues form a β-sheet conformation It is more likely, and the amino acid residues at positions 9-21 may also form a β-sheet conformation. The β-sheet conformation favors aggregation of the Aβ _1-42 peptide. The experimental results showed that the three amino acid residues Val ⁴⁰ Ile ⁴¹ Ala ⁴² at the C-terminal stabilized the conformation of the β-sheet, which was beneficial to the formation of the β-sheet. The N-terminus of Aβ peptide _1-42 is hydrophilic and can form an α-helix, random helix or β-sheet conformation depending on the solution conditions. The results of the study indicated that the β-sheet conformation was favorable for the aggregation of Aβ peptides, and the aggregation of Aβ peptides was due to the interaction of its hydrophobic regions. Soto et al. (Soto C, Kindy MS, Baumann M, et al., Inhibition of Alzheimer's amyloidosis by peptides that prevent beta-sheet conformation. Biochem. Biophys. Res. Commun. 1996, 226(3): 672-680) Substituting proline for amino acids near the hydrophobic region of the Aβ peptide, the resulting small peptide not only does not form a β-sheet conformation, but can also bind to the Aβ peptide to maintain a random helical conformation and inhibit its aggregation. The results of the study found that one of the hydrophobic regions of the Aβ peptide: the Aβ _16-20 pentapeptide fragment Lys-Leu-Val-Phe-Phe can bind to the Aβ peptide, thereby preventing its aggregation. Substitution by alanine one by one indicated that Lys ¹⁶ , Leu ¹⁷ , and Phe ²⁰ played a key role therein. This indicates that ^Aβ16-20 residues are the sites where Aβ peptides bind to adjacent Aβ peptide chains during Aβ peptide aggregation. Studies have shown that the spatial conformation of Aβ significantly affects its aggregation ability. When its secondary structure is dominated by α-helices, the aggregation is slow; while when the secondary structure is dominated by β-sheets, the aggregation is faster.

Under certain conditions, the exposure of the hydrophobic region of the structure rich in β-sheets will promote the aggregation of Aβ, form oligomers, and finally become insoluble substances deposited in the interneuron gap, resulting in neurotoxicity and increased activity of glial cells in the brain. Inflammatory mediators and complement together form amyloid plaques.

The increase in the number of hydrophobic charges of molecules is one of the main reasons for Aβ aggregation. Compared with Aβ _1-40 , the extended two amino acids of Aβ _1-42 not only increase the hydrophobicity of Aβ, making it easier to aggregate, but also improve the stability of aggregates, which can be selectively present in amyloid plaques at an early stage deposition. Aβ _1-42 may be an initiating factor for soluble Aβ to form oligomers, fibers and plaques (Younkin, SG1995. Evidence that A beta 42 is the real culprit in Alzheimer's disease. Ann. Neurol. 37: 287-288.; Matsuok Y, Saito M, Lafrancois T, et al. Noval therapeutic approach for the treatment of Alzheimer's disease by peripheral administration of agents with an affinity to β-amyloid [J]. J Neurosci, 2003, 23(1): 1-5).

Jarrett et al. proposed that: Aβ _1-42 first aggregated as a "seed" to initiate the deposition of Aβ; other monomers gradually aggregated around the nucleus, elongated (elongation) peptide chains formed fibers to further aggregate and spread, and finally formed plaques (Jarrett et al. JT, Lansbury PT Jr. Seeding "one-dimensional crystallization" of amyloid: a pathogenic mechanism in Alzheimer's disease and scrapie? Cell. 1993, (6): 1055-1058).

Aβ aggregation, precipitation and formation of senile plaques and the accompanying neuronal damage are considered to be the central link in the pathological mechanism of Alzheimer's disease. Therefore, if the aggregation of Aβ peptide can be inhibited and the degradation and clearance of Aβ peptide can be accelerated, the purpose of preventing and/or treating AD can be fundamentally achieved.

At present, the research goals of drugs against Aβ in the world are to reduce the generation of Aβ, increase the clearance of Aβ, prevent or reverse the aggregation of Aβ, and inhibit the toxicity of Aβ. After removing amyloid plaques from the brains of mice with Alzheimer's disease, the mice's brain cells began to miraculously regain function, researchers at Washington University School of Medicine found. It shows that drugs targeting Aβ have encouraging prospects. Among various drugs targeting Aβ, β-sheet blockers are attracting more and more attention.

At present, there are mainly two types of drugs that belong to β-sheet blockers in the world:

(1) Based on the discovery that low molecular weight aminoglycoproteins (gly-cosaminoglycan, GAGs) play a role in stabilizing plaques and hindering plaque degradation in β-amyloid plaques, the Canadian company Neurochem designed and synthesized the GAGs derivative compounds. Experiments in animals have shown that such low molecular weight GAG analogs can significantly reduce the level of β-amyloid in plasma and brain, inhibit Aβ aggregation, and be used for the treatment of AD. Currently, the small molecule compound Alzheimer is in phase III clinical trials.

(2) Chacón et al. (Chacón MA, Barría MI, Soto C, et aL., Beta-sheetbreaker peptide prevents beta-induced spatial memory impairments with partial reduction of amyloid deposits. Mol. Psychiatry. 2004, 9(10): 953-61 ) induced behavioral disorder in rats by intraperitoneal injection of fibrotic Aβ, and then tested the protective effect of fiye-amino-acid beta-sheet breaker peptide (iAbeta5p) on neurons , the results show that iAbeta5p can not only prevent the formation of Aβ fibers, but also has the ability to decompose Aβ fibers. The drug is currently in phase III clinical trials.

The field currently needs to be able to specifically bind to β-amyloid monomers (Aβ _1-42 ), stabilize its normal spatial structure, inhibit its formation of β-sheets, and prevent soluble β-amyloid oligomers and β-amyloid plaques Formation of new active agents. The medicament should be able to inhibit the aggregation of Aβ peptide and accelerate the degradation and clearance of Aβ peptide, so that it can be used for the prevention and/or treatment of AD.

Contents of the invention

An object of the present invention is to provide a method that can specifically bind to β-amyloid monomers (Aβ _1-42 ), stabilize its normal spatial structure, inhibit its formation of β-sheets, prevent soluble β-amyloid oligomers and β- A polypeptide that forms amyloid plaques and has a decomposing effect on Aβ fibrils may be called a β-sheet blocking peptide.

The inventors of the present invention unexpectedly found that a polypeptide comprising the following amino acid sequence can achieve the above object:

His-X1-X2-Leu-X3-Phe-Phe-X4-Glu-Asp

Wherein: X1 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

X2 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

X3 can be a valine residue (Val, V) or a proline residue (Pro, P);

X4 can be an alanine residue (Ala, A) or a glutamic acid residue (Glu, G).

Therefore, in a first aspect of the present invention, there is provided a polypeptide comprising the above amino acid sequence.

Since the above polypeptides can specifically bind to β-amyloid monomers (Aβ _1-42 ), stabilize its normal spatial structure, inhibit its formation of β-sheets, and prevent soluble β-amyloid oligomers and β-amyloid plaques It can form and decompose Aβ fibers, so it can be used for the prevention and/or treatment of Alzheimer's disease.

Therefore, in the second aspect of the present invention, the use of the polypeptide of the present invention in the preparation of a medicament for preventing and/or treating Alzheimer's disease is provided.

In the third aspect of the present invention, a pharmaceutical composition is provided, which comprises one or more polypeptides of the present invention and a pharmaceutically acceptable carrier.

In the fourth aspect of the present invention, there is provided a method for preventing and/or treating Alzheimer's disease in a subject, the method comprising administering to the subject an effective amount of the polypeptide of the present invention.

Description of drawings

Figure 1 shows the steps of an exemplary method for preparing the polypeptide of the present invention and the test results of the prepared product. Figure 1A shows the main steps of an exemplary method for synthesizing and purifying the polypeptide H101 of the present invention; Figure 1B shows the chromatographic analysis results of H101; Figure 1C shows the mass spectrometric detection results of H101.

Figure 2 shows the results of Thioflavin T fluorescence analysis of Aβ _1-42 peptide aggregation and fiber formation, indicating the effect of the polypeptide of the present invention on the fluorescence intensity of Aβ aggregation. Aβ _1-42 peptide at a given concentration was incubated with polypeptide H101, H102 or H103 of the present invention or vitamin E (VE) or L5 at 37° C. for 24 hours, and then ThT fluorescence intensity was measured. n=5, * indicates P<0.05 compared with Aβ _1-42 group.

Figure 3 shows the dose-dependent curves of H102 and vitamin E inhibiting Aβ _1-42 fibril formation. Aβ _1-42 were co-incubated at 37 °C for 24 hr, and then ThT fluorescence intensity was measured. n=5.

Figure 4 shows the time-dependent curves of the inhibition of _Aβ1-42 fibril formation by H102 and vitamin E. H102 and vitamin E at a concentration of 44.30 μmol/L were co-incubated with 11.07 μmol/L _Aβ1-42 at 37 °C, respectively. ThT fluorescence intensity was measured at 12 hours, 1 day, 3 days, 5 days and 7 days. n=5.

Fig. 5 shows the electron microscope observation results of Aβ _1-42 polypeptide fiber formation after incubation with various polypeptides at a concentration of 44.30 μmol/L and 11.07 μmol/L Aβ _1-42 at 37°C for 5 days. Among them: Figure 5A is the electron microscope result of Aβ _1-42 incubated alone for 5 days, with a magnification of 35,000 times; Figure 5B is the electron microscope result of Aβ _1-42 incubated alone for 5 days, with a magnification of 50,000 times; Figure 5C is the result of the polypeptide L5 The electron microscope result of incubation with Aβ _1-42 for 5 days, the magnification is 35000 times; Figure 5D is the electron microscope result of incubation of polypeptide H101 with Aβ _1-42 for 5 days, the magnification is 35000 times; _1-42 were incubated together for 5 days, with a magnification of 35,000 times; Figure 5F is the electron microscope result of polypeptide H103 incubated with Aβ _1-42 for 5 days, with a magnification of 35,000 times;

Figure 6 shows the survival of various polypeptides at different concentrations (10 μmol/L, 20 μmol/L and 40 μmol/L) for human neuroblastoma SH-SY5Y cells cultured with 5 μmol/L of Aβ _1-42 for 72 hours rate impact. n=12, * indicates P<0.05 compared with Aβ _1-42 group.

Figure 7 shows the results of immunohistochemical staining of neuron sections in the CA1 region of the hippocampus of the mice in each group using the APPN terminal antibody and the Aβ antibody, with a magnification of 400 times. Figure 7A: Immunohistochemical staining results of APP N-terminal antibody in the control group; Figure 7B: Immunohistochemical staining results of Aβ antibody in the control group; Figure 7C: Immunohistochemical staining results of APP N-terminal antibody in the model group; Figure 7D: Model Aβ antibody immunohistochemical staining results of the polypeptide group; Figure 7E: APP N-terminal antibody immunohistochemical staining results of the polypeptide injection group; Figure 7F: Aβ antibody immunohistochemical staining results of the polypeptide injection group.

Figure 8 shows the results of staining the temporal lobe cortex and hippocampus of animals in each group with Congo red, the magnification is 400 times. Figure 8A: Congo red staining results of the brain temporal lobe cortex of the control group; Figure 8B: Congo red staining results of the hippocampus of the control group; Figure 8C: Congo red staining results of the brain temporal lobe cortex of the model group; Figure 8D: Model group Congo red staining results of the hippocampus; FIG. 8E : Congo red staining results of the temporal lobe cortex of the polypeptide injection group; FIG. 8F : Congo red staining results of the hippocampus of the polypeptide injection group.

Detailed ways

The amino acid sequences of a series of polypeptides are disclosed herein, and those skilled in the art can understand that when a sequence is represented by single-letter or three-letter amino acid residues, the sequence represents from left to right the polypeptide from The sequence from the N-terminus (amino-terminus) to the C-terminus (carboxyl-terminus). For example, when "His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp" or "HQKLVFFAED" is used to indicate the sequence of a polypeptide, it means that the sequence of the polypeptide is "N-terminal-His- Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp-C-terminus", that is, "N-terminus-HQKLVFFAED-C-terminus".

In a first aspect of the present invention, a polypeptide comprising the following amino acid sequence is provided:

His-X1-X2-Leu-X3-Phe-Phe-X4-Glu-Asp

Wherein: X1 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

X2 can be a lysine residue (Lys, K) or a glutamine residue (Gln, Q);

X3 can be a valine residue (Val, V) or a proline residue (Pro, P);

X4 can be an alanine residue (Ala, A) or a glutamic acid residue (Glu, G).

The polypeptide of the present invention may comprise, consist essentially of, or consist of the above amino acid sequence.

In one embodiment of the present invention, there is provided a polypeptide having the following sequence, which is named H101 in the present invention:

H101: His-Lys-Gln-Leu-Val-Phe-Phe-Glu-Glu-Asp (HKQLVFFEED) (SEQ ID NO: 1).

In another embodiment of the present invention, there is provided a polypeptide having the following sequence, which is named H102 in the present invention:

H102: His-Lys-Gln-Leu-Pro-Phe-Phe-Glu-Glu-Asp (HKQLPFFEED) (SEQ ID NO: 2).

In another embodiment of the present invention, there is provided a polypeptide having the following sequence, which is named H103 in the present invention:

H103: His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp (HQKLVFFAED) (SEQ ID NO: 3).

The amino acid sequences of various polypeptides are disclosed herein. It will be apparent that various modifications can be made to the polypeptides of the invention. Such modifications include, but are not limited to: hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, lysine, arginine and histidine side chains methylation of the o-amino group, acetylation of the N-terminal amino group and in some cases amidation of the C-terminal carboxyl group. It should be understood that after the amino acid sequence of the polypeptide of the present invention is known, the various modifications mentioned above will be obvious to those skilled in the art, and polypeptides containing these modifications including the disclosed amino acid sequence are also within the scope of the present invention.

The above-mentioned polypeptides of the present invention can be used as β-sheet blocking peptides, and they can specifically bind to β-amyloid monomers (Aβ _1-42 ), stabilize its normal spatial structure, inhibit its formation of β-sheets, and prevent soluble β-amyloid Protein oligomers and β-amyloid plaques form and have a decomposing effect on Aβ fibers, so they can be used as a drug for preventing and/or treating Alzheimer's disease.

Therefore, in another aspect of the present invention, the use of the polypeptide of the present invention in the preparation of a drug for preventing and/or treating Alzheimer's disease is provided.

As used herein, "prevention" refers to reducing a subject's risk of developing a disease or delaying the onset of disease or symptoms in a patient. "Treatment" as used herein does not mean complete cure. It refers to the alleviation of the symptoms of the underlying disease and/or the reduction of one or more underlying cellular, physiological or biochemical etiologies or mechanisms causing the symptoms. It should be understood that alleviated as used herein is relative to a disease state and includes the molecular state of the disease, not just the physiological state of the disease.

The present invention also provides a pharmaceutical composition, which includes one or more polypeptides of the present invention and a pharmaceutically acceptable carrier.

"Pharmaceutically acceptable carrier" refers to a substance that will not have adverse effects biologically or otherwise, that is, the substance can be administered to a subject together with the polypeptide of the present invention without causing any adverse biological effects. affect or interact in a deleterious manner with any other component of the pharmaceutical composition in which it is contained. It is obvious that the carrier should be chosen to minimize any degradation of the active ingredient and minimize any adverse side effects in the subject, as is well known to those skilled in the art.

The pharmaceutical compositions of the present invention generally include at least one polypeptide of the present invention and one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to: antioxidants, preservatives, colorants, flavorants and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, carriers, diluents, excipients and / or pharmaceutical adjuvants. For example, suitable carriers may be physiological saline solution, citrate buffer, or artificial CSF, possibly with the addition of other substances commonly used in parenteral compositions. Neutral buffered saline or saline mixed with serum albumin are also exemplary carriers. Those skilled in the art can readily determine a variety of buffers that are useful in the compositions and dosage forms of the present invention. Typical buffering agents include, but are not limited to, pharmaceutically acceptable weak acids, weak bases or mixtures thereof. Preferably, the buffering component is a water-soluble substance such as phosphoric acid, tartaric acid, lactic acid, succinic acid, citric acid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, and salts thereof.

The base solvent in the vehicle can be aqueous or non-aqueous in nature. In addition the carrier may contain other pharmaceutically acceptable excipients for improving or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, dissolution rate or odor of the formulation. The pharmaceutical composition of the present invention may also contain other pharmaceutically acceptable carriers for improving or maintaining the release rate of the polypeptide of the present invention. Such carriers are materials known to those skilled in the formulation of sustained release formulations.

When the pharmaceutical composition of the present invention is formulated, it can be stored in sterile tubes in the form of solutions, suspensions, gels, emulsions, solids or dehydrated or lyophilized powders. These formulations may also be stored in ready-to-use form, as a lyophilized powder to be reconstituted before use, or as a liquid to be diluted before use. Preferably, the pharmaceutical composition of the present invention is provided in a single-use sterile tube and stored at 2-8°C until use. Immediately before administration, the pharmaceutical compositions of the present invention may be suitably diluted with a suitable sterile citrate buffer, eg, any of the above mentioned citrate buffers.

The present invention also provides a method for preventing and/or treating Alzheimer's disease in a subject, the method comprising administering to the subject an effective amount of the polypeptide of the present invention.

The term "effective amount" means that the amount of the compound used is sufficient to prevent the onset or symptoms of a disease, or to ameliorate one or more causes or symptoms of a disease or disorder. The improvements need only be mitigated or altered and not necessarily eliminated. For the polypeptides of the present invention, the prophylactically and/or therapeutically effective amount will vary depending on the individual targeted and the polypeptide used. The prophylactically and/or therapeutically effective amount of polypeptide used will also vary according to the age, size, weight, condition, etc. of the individual. It is within the ability of those skilled in the art to determine a prophylactically and/or therapeutically effective amount of a polypeptide of the invention for a particular subject.

When the polypeptide of the present invention or the prevention and/or treatment method of the present invention is applied to a subject with Alzheimer's disease, it can significantly inhibit the aggregation of Aβ in the brain tissue of the subject, reduce the number and area of amyloid plaques, improve The effect of Alzheimer's disease symptoms. For example, it can improve activity and concentration and reduce reaction time, and can improve pronunciation, facial expression, posture, sense of smell, libido, sexual function and emotional status and improve mental state. In another embodiment of the present invention, the polypeptide of the present invention can be suitably administered to, for example, patients with Alzheimer's disease as a cognition enhancing agent, thereby improving learning ability especially impaired by dementia or inhibiting cognitive decline and/or dementia.

Preparation of the polypeptide of the present invention

The polypeptides of the present invention can be prepared by any method known to those skilled in the art for preparing polypeptides.

Polypeptides of the invention can be synthesized using chemical synthesis. Polypeptide synthesis can be performed in solution or using solid-phase synthesis. The solid-phase synthesis method of polypeptide includes Fmoc solid-phase synthesis method and tBoc solid-phase synthesis method. The method of artificially synthesizing polypeptides is generally synthesized from the C-terminus (carboxyl-terminus) to the N-terminus (amino-terminus).

In one embodiment of the present invention, the polypeptide of the present invention is synthesized by Fmoc solid-phase synthesis method, and purified by HPLC. Fig. 1 shows in an exemplary manner the main steps of synthesizing and purifying a polypeptide H101 of the present invention and the detection results of the prepared polypeptide in this embodiment. In this embodiment, a polypeptide solid-state synthesizer is used to synthesize the polypeptide on a synthesis column, thereby greatly reducing the difficulty of product purification. To prevent side reactions, the synthesis columns and the side chains of the added amino acids are protected. The carboxyl terminus is free and must be activated prior to the reaction. The polypeptide of the present invention can be identified by mass spectrometry (MS) analysis after being synthesized by Fmoc method and purified by preparative high performance liquid phase (HPLC) column. It should be understood that all polypeptides of the present invention can be prepared, purified and identified using synthetic methods similar to the above embodiments.

The polypeptide of the present invention can also be produced by recombinant genetic engineering. In short, the polynucleotide encoding the polypeptide of the present invention can be synthesized, then transformed into a suitable host cell and expressed by methods known in the art, and the expression product can be purified or processed to obtain the present invention. Invented peptides.

Example

The present invention will be further described below in conjunction with embodiment. The following examples are intended to enable those skilled in the art to better understand the present invention, which are for illustrative purposes only and are not intended to limit the scope of the present invention. Efforts have been made to ensure accuracy with respect to numbers (eg, amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C or is at ambient temperature, and pressure is near or at atmospheric.

Embodiment 1: the preparation of polypeptide of the present invention

In this example, a polypeptide H101 of the present invention was first synthesized using the Fmoc/tBu solid-phase polypeptide synthesis method, and the sequence of the polypeptide is:

His-Lys-Gln-Leu-Val-Phe-Phe-Glu-Glu-Asp (SEQ ID NO: 1).

The raw materials used in the polypeptide synthesis method of this embodiment are: Fmoc-Asp(OtBu)-Wang Resin(0.37mmol/g), Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-His(trt)- OH, Fmoc-Gln(trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH and Fmoc-Phe-OH.

The main steps of the peptide synthesis and purification method used in this example are shown in Figure 1A.

The formula of TFA reagent used in the experimental scheme shown in Fig. 1A is: [TFA:H ₂ O:ethanedithiol:phenol, mixed in a volume ratio of 92.5:2.5:2.5:2.5].

The prepared crude peptide product was purified by HPLC, so that the purity of the finally obtained peptide was greater than 95%. The HPLC results are shown in FIG. 1B .

The polypeptide H101 prepared and purified by the above method was identified and sequenced using mass spectrometry (MS) analysis, and the results of mass spectrometry analysis are shown in Figure 1C. The results of identification and sequencing showed that the sequence and molecular weight of the polypeptide prepared by the above method were consistent with expectations.

Using a method similar to that in this example, the following polypeptides of the present invention were also prepared subsequently:

H102: His-Lys-Gln-Leu-Pro-Phe-Phe-Glu-Glu-Asp (SEQ ID NO: 2);

H103: His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp (SEQ ID NO: 3).

The results of sequencing and identification of the above several synthetic polypeptides show that the synthesis and purification methods described in this example are applicable to various polypeptides of the present invention. The sequences and molecular weights of various polypeptides prepared by the method are consistent with expectations. And the polypeptide product with a purity greater than 95% can be obtained by the above-mentioned purification method.

Embodiment 2: The effect of the polypeptide of the present invention on β-amyloid protein

Materials and Methods

1. Drugs and reagents: Aβ _1-42 (purity>98%), vitamin E, Thioflayin T (Thioflayin T, ThT), thiazolium blue (MTT), dimethyl sulfoxide (DMSO), all purchased from Sigma, USA Company; MEM culture medium, fetal bovine serum (FBS), trypsin (trypsin) are all purchased from U.S. Gibco-BRL company; Several polypeptides H101, H102 and H103 of the present invention are prepared by the method described in Example 1; A pentapeptide Leu-Pro-Phe-Phe-Asp was synthesized by a method similar to that described in Example 1, which has the same sequence as the beta-sheet blocking peptide iAbeta5 invented by Soto-Jara et al., and Named L5 in the present invention, the L5 peptide will be used as a control in this example and the following examples. The above peptides were all synthesized by Shanghai Jill Biological Co., Ltd., and purified by high performance liquid chromatography (High performance Liquid Chromatography, HPLC), and the purity was >95% identified by mass spectrometer (MS).

2. Thioflavin T (ThT) fluorescence analysis and electron microscope observation: ThT fluorescence intensity can reflect the aggregation degree of Aβ, so ThT fluorescence is used to analyze the aggregation degree of the protein. It has been reported that vitamin E (VE) can effectively inhibit Aβ aggregation and fibril formation, so vitamin E was used as a positive control in this experiment. The Aβ _1-42 freeze-dried powder was prepared into a 22.15 μmol/L solution with 50 mmol/L PBS (pH7.4), and the above four polypeptides (H101, H102, H103 and L5) were prepared with the same PBS to a concentration of 88.61 μmol/L solution, vitamin E was prepared into 88.61μmol/L emulsion with PBS containing 0.2% Tween 80. During the experiment, the Aβ _1-42 solution was mixed with various polypeptide solutions in equal volumes, so that their final concentrations were 11.07 μmol/L and 44.30 μmol/L, respectively. Aβ _1-42 and vitamin E emulsion were mixed in equal volumes as a positive control, and a solution containing only Aβ _1-42 without any of the above polypeptides or vitamin E was used as a negative control. After incubating the solutions of the above groups at 37°C for 24 hours, 10 μl of each group was added to 990 μl of 3.0 μmol/L ThT phosphate buffer, and the fluorescence intensity of ThT was measured at an excitation wavelength of 453 nm and an emission wavelength of 478-486 nm.

3. The dose-effect and time-effect relationship between the polypeptide of the present invention and Aβ _1-42 : select the polypeptide H102, prepare a series of concentrations with the above-mentioned PBS, and incubate with 11.07 μmol/L Aβ _1-42 solution at 37°C for 24 hours and then take a sample for ThT Fluorescence detection is used to determine the dose-effect relationship between the polypeptide of the present invention and Aβ _1-42 . H102 and vitamin E at a concentration of 44.30 μmol/L were co-incubated with 11.07 μmol/L Aβ _1-42 at 37°C for 12 hours (12h), 1 day (1d), and 3 days (3d) after the start of incubation, respectively. , 5 days (5d) and 7 days (7d) samples were taken for ThT fluorescence detection to determine the time-dependent relationship between various peptides and Aβ _1-42 . At the same time, Aβ _1-42 peptide was incubated alone as a negative control.

4. Electron microscope observation: In order to further observe the formation of fibers, incubate with various peptides at a concentration of 44.30 μmol/L and 11.07 μmol/L Aβ _1-42 at 37°C for 5 days, then take 5 μl of each group of samples and drop them on The 300-mesh deionized copper grid containing the carbon support film was allowed to stand at room temperature for 15 minutes. Negative stain with 2% uranyl acetate in the dark for 2 minutes, and observe with a transmission electron microscope after drying. At the same time, the Aβ _1-42 peptide was incubated alone for 5 days as a negative control.

5. Cytotoxicity experiment: the commonly used human neuroblastoma SH-SY5Y cell line in the field was selected as the object of cytotoxicity experiment. Add 10% (v/v) fetal calf serum, culture at 37°C, 5% CO ₂ incubator, and pass passage once every 5 days. Inoculate SH-SY5Y cells in a stable growth state and in the logarithmic growth phase in a 96-well plate (Costar product) at a density of 1.0×10 ⁴ /ml, with a culture medium volume of 200 μl in each well, and replace it with serum-free culture after 24 hours base, and added 5 μmol/L Aβ _1-42 solution, grouped. The aforementioned four polypeptides were added to the culture medium of each group of cells at concentrations of 10 μmol/L, 20 μmol/L and 40 μmol/L, respectively. The cell group in which only serum-free medium was added without Aβ _1-42 and polypeptide was used as a positive control, and the cell group in which only Aβ _1-42 was added to the medium without the above polypeptide was used as a negative control. 72 hours after adding the polypeptide, 20 μl of 3-(4,5-dimethyl-2-thiazole)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/ml) was added to each well, 37 Incubate at ℃ for 4 hours, discard the original culture medium, add 200 μl of DMSO to each well, let stand for 10 minutes, shake for 1 minute to completely dissolve the formazan particles, and measure the optical density (Optical density, OD) at 492 nm with an automatic microplate reader .

6. Statistical processing: For the data comparison between two groups, the t test method is used; for the data comparison of more than two groups, the F test method is used.

result

1. Inhibition of Aβ _1-42 Aggregation and Fibril Formation by Various Peptides Tested

The effects of various peptides and VE tested on Aβ _1-42 aggregation and fiber formation were analyzed by ThT fluorescence method. Aβ _1-42 can form a very high fluorescence intensity in PBS solution, indicating that Aβ _1-42 can self-aggregate and Aβ fibrils are formed. The fluorescence intensity of the negative control containing A[beta _]1-42 alone without any test polypeptide or vitamin E was counted as 100%. The inhibition rate (%) of Aβ _1-42 aggregation and fibril formation was calculated for each polypeptide tested. The results are shown in Table 1 below (note: the background fluorescence generated by the instrument system itself has been deducted for each data in Table 1).

Table 1: Inhibitory effect of various polypeptides on Aβ aggregation (%)

Peptides tested L5 H101 H102 H103 Vitamin E Inhibition rate) 25.74 24.79 27.84 14.91 24.85

From the results in Table 1 above, it can be seen that H102 has the highest inhibitory rate on Aβ _1-42 aggregation, and the descending order is: L5, vitamin E, H101 and H103.

The experimental results in the above table are drawn into a histogram, which is shown in Figure 2.

As shown in Figure 2, H102 can significantly inhibit the aggregation and fiber formation of Aβ _1-42 ; co-incubation with vitamin E and Aβ _1-42 can also significantly inhibit the aggregation and fiber formation of Aβ _1-42 , but the effect is weaker than that of H102.

2. The dose-effect relationship between the polypeptide of the present invention and Aβ _1-42 : Figure 3 shows the ThT fluorescence after co-incubating with 11.07 μmol/L Aβ _1-42 solution of different concentrations of polypeptide H102 and vitamin E at 37°C for 24 hours The result of the test. It can be seen from Figure 3 that H102 and vitamin E have a concentration-dependent effect on the inhibition of Aβ fibril formation, and the inhibition rate of H102 at each concentration point is greater than that of vitamin E. The highest inhibitory concentrations of H102 and vitamin E were around 20μmol/L.

3. The time-dependent relationship between the polypeptide of the present invention and Aβ _1-42 : Figure 4 shows the results of ThT fluorescence detection after co-incubating the polypeptide H102 of the present invention and vitamin E with 11.07 μmol/L Aβ _1-42 solution at 37°C for different times. result. It can be seen from Figure 4 that the fluorescence intensity of 11.07 μmol/L Aβ _1-42 was low before 24 hours of incubation alone, and increased sharply on the 3rd day, and then formed a plateau growth, the fluorescence intensity of each time point in the H102 group and the VE group All significantly weakened, and the effect of the H102 treatment group was the most significant. The time T _1/2 of the half peak of the curve represents the change characteristics of the curve, and the T _1/2 of the Aβ group is on the third day. Compared with the Aβ group, the H102 and vitamin E group delayed T _1/2 to day 4.

4. Electron microscope observation results:

Fig. 5 shows the electron microscope observation results of Aβ _1-42 polypeptide fiber formation after incubation with various polypeptides at a concentration of 44.30 μmol/L and 11.07 μmol/L Aβ _1-42 at 37°C for 5 days. It can be seen from each figure that after Aβ _1-42 was incubated alone for 5 days, a large number of Aβ fibers appeared, with a diameter of about 8-10nm and a length of about 0.5-5μm, which were aggregated in the form of prickly crystals, branched, and even interwoven into a network , interspersed with a small amount of aggregated amorphous structure (Figure 5A and 5B, control group); L5 was incubated with Aβ for 5 days, and the formed Aβ fibers were obviously thinner, and aggregated into a diameter of about 4-gnm and a length of 0.3-5 μm, Intertwined into a network, interspersed with a very small amount of aggregated amorphous structure (Fig. 5C); H101 and Aβ were co-incubated for 5 days, and Aβ fibers were formed, with a diameter of 5-8 nm and a length of 0.5-5 μm. The branches interweave into a network, and a very small amount of aggregated amorphous structure can be seen among them (Fig. 5D); H102 and Aβ were co-incubated for 5 days, and the formation of Aβ fibers was significantly reduced and the diameter became thinner, about 3-6 nm, 0.3-3 μm in length, interweaving form a network, interspersed with a very small amount of aggregated amorphous structure (Figure 5E); H103 and Aβ were co-incubated for 5 days, Aβ fibers formed, and aggregated into a diameter of about 5-8nm, a length of 0.5-5μm, in the form of prickles The crystals were aggregated and interwoven into a network, interspersed with a small amount of amorphous structure in aggregated state (Fig. 5F).

5. The effect of the polypeptide of the present invention on the survival rate of SH-SY5Y cells

Figure 6 shows the survival rate of various polypeptides at different concentrations (10 μmol/L, 20 μmol/L and 40 μmol/L) for human neuroblastoma SH-SY5Y cells cultured with 5 μmol/L of Aβ _1-42 for 72 hours Impact. n=12, * indicates P<0.05 compared with Aβ _1-42 group.

It can be seen from Figure 6 that after Aβ _1-42 acted on SH-SY5Y cells for 72 hours, the cell viability decreased, while four kinds of polypeptides with different concentrations (10μmol/L, 20μmol/L, 40μmol/L) were used to treat Aβ _1-42 co-incubated cells, the cell survival rate improved, and showed a certain dose-dependent relationship. The above results indicated that H102 was the most effective in increasing cell viability, and the optimal concentration was 20 μmol/L.

Example 3: H102 on amyloid (Aβ) and amyloid pro-amyloid in the brain of APP transgenic mice The effect of body protein (APP) expression

Materials and Methods

Experimental animals: 30 9-month-old APP 695 transgenic mice, 10 C57BL/6J mice of the same genetic background and the same month age, male or female, were purchased from the Experimental Animal Research Center of Chinese Academy of Medical Sciences and Peking Union Medical College.

Drugs and reagents: Aβ _1-42 antibody was purchased from Chemicon, USA; APP antibody, ready-to-use SABC immunohistochemical staining kit, DBA chromogenic kit, and Congo red were purchased from Wuhan Boster Company; other reagents were domestically produced for analysis pure. The H102 polypeptide was prepared, purified and identified using the method described in Example 1, and was diluted with physiological saline to prepare a 40 μmol/L solution during the experiment.

Animal grouping and model preparation: 30 APP 695 transgenic mice were randomly divided into a model group of 15 and a polypeptide injection group of 15; 10 C57BL/6J mice were set as the normal control group. Polypeptide injection group: inject 3 μl of 40 μmol/L H102 normal saline solution into the lateral ventricle of the animals each time, control group and model group: inject the same volume of normal saline into the lateral ventricle of the animals each time, once a day, and continuously inject for 10 days. All animals were kept under the same conditions in SPF-grade clean rat rooms in the Experimental Animal Center of Tianjin Medical University.

After the animal was anesthetized with 10% chloral hydrate at a dose of 400 mg/Kg body weight, it was fixed on the brain stereotaxic instrument, the hair on the head was cut off, and after being disinfected with ammonium iodine, the skin length of the head was cut along the midline. About 1cm, peel off the periosteum, revealing bony landmarks. Adjust the plane of the mouse head so that Bregma and Lambda are at the same height. Referring to George's mouse brain stereotaxic atlas, a small hole with a diameter of 1 mm was drilled on the skull at the position of the lateral ventricle (P _0.58 , R ₁ , H _-1.5 ) as the needle entry point. The day after surgery, the injections were started. Slowly and uniformly inject 3 μl of polypeptide solution or normal saline into the lateral ventricle through a microsyringe with the help of a small animal brain stereotaxic instrument. After the injection, the needle is left in place for 1 min, and the needle is withdrawn slowly. Local compression to stop bleeding, continuous injection for 10 days.

Preparation of paraffin sections of mouse hippocampus: After 10 days of continuous intracerebroventricular injection of animals in each group, 0.1kg/L chloral hydrate was anesthetized by intraperitoneal injection at a dose of 4mL/kg body weight, and the left ventricle was quickly intubated. Open the right atrium, and quickly perfuse normal saline; after the liver turns white, change the perfusion to 40g/L paraformaldehyde, the speed is first fast and then slow, until the limbs of the mouse are stiff; take the mouse brain, and quickly put it into 30% sucrose fixed in paraformaldehyde solution for 2-4 days. After the brain tissue sinks, it can be embedded in wax block. The sagittal section of the mouse brain was performed, and after the CA1 region of the hippocampus appeared, serial sections were taken, and one slice was taken every 10 slices, and three slices were taken for each antibody for each mouse, with a thickness of 5 μm.

Immunohistochemical staining: According to the steps in the instructions of the immunohistochemical kit, the immunohistochemical staining of Aβ and APP were carried out respectively, the dilution ratio of the first antibody was 1:100 and 1:200, and the dilution ratio of the second antibody was 1: 100. Observe and photograph at 400x magnification.

Congo red staining of mouse brain tissue sections: the above paraffin-embedded sections were dewaxed to water, immersed in 10% formaldehyde solution for 15 minutes, directly immersed in Congo red staining solution for 15 minutes, washed in water for 4 minutes, immersed in hematoxylin solution for 2 minutes, and immersed in 0.5 % hydrochloric acid ethanol differentiation, fully washed with water, dehydrated with ethanol, transparentized with xylene, and mounted with neutral gum. Observe and photograph at 400x magnification.

result

1. APP N-terminal antibody immunohistochemical staining and Aβ antibody immunohistochemical staining.

Figure 7 shows the results of immunohistochemical staining of neuronal sections in the hippocampal CA1 region of animals in each group with APP N-terminal antibody and Aβ antibody.

It can be seen from Figure 7: (1) The results of immunohistochemical staining of APP N-terminal antibody and Aβ antibody in the control group (Figure 7A and Figure 7B respectively) showed that the cytoplasmic staining of neurons in the CA1 region of the hippocampus in the control group was negative or weakly positive; (2) The results of immunostaining of APP N-terminal antibody and Aβ antibody in the model group (Fig. The staining was obviously deepened; (3) The results of immunohistochemical staining of APP N-terminal antibody and Aβ antibody in the polypeptide injection group (Figure 7E and Figure 7F respectively) showed that compared with the model group, APP positive cells in the polypeptide injection group decreased, The coloring fades and the expression weakens.

2. Congo red staining.

After staining the mouse brain pathological sections with Congo red, the expression of amyloid plaques in the temporal lobe cortex and hippocampus of the mouse brain was observed, and the staining results are shown in FIG. 8 .

It can be seen from Figure 8 that: (1) no positive amyloid plaques were seen in the temporal lobe cortex (Figure 8A) and hippocampus (Figure 8B) of the mice in the control group; (2) the temporal lobe of the mice in the model group The cortex (Fig. 8C) and hippocampus (Fig. 8D) appeared in different sizes, light red stained lumps of amyloid plaques, most of which were round or almost round, scattered and uneven in distribution density, and how many were seen around. (3) The brain temporal lobe cortex (Fig. 8E) and hippocampus (Fig. 8F) of the mice in the polypeptide injection group also had some agglomerated amyloid plaque substances, but the amyloid plaques in the polypeptide injection group The number of plaques was significantly less than that of the model group.

in conclusion

The results of immunohistochemical staining showed that in the CA1 area of the brain of the mice in the model group, the number of Aβ _1-42 positive cells was significantly higher than that in the normal control group. The number of positive cells in the H102 polypeptide injection group was significantly less than that in the model group. Therefore, it is speculated that H102 can inhibit the aggregation of Aβ in vivo and reduce the toxicity of Aβ. In addition, Congo red staining found that amyloid plaques appeared in the brains of mice in the transgenic group, but no plaques were seen in the brains of mice in the normal control group. The number and area of amyloid plaques in the brains of mice in the H102 polypeptide injection group There was a marked reduction in the transgenic group. This also verified the conclusion that H102 can inhibit the aggregation of Aβ and reduce its generation.

Throughout this application, various publications are cited. The publications mentioned are incorporated by reference in their entirety into this specification in order to more fully describe the state of the art to which this invention pertains.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope and spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and examples of the invention disclosed herein. The specification and examples should be considered for illustrative purposes only, with the true scope and spirit of the invention set forth in the appended claims.

sequence listing

<110> Tianjin Medical University

Wang Wei

<120> β-sheet blocking peptide for preventing and/or treating Alzheimer's disease

<130>CP1080054/CB

<160>4

<170>PatentIn version 3.3

<210>1

<211>10

<212>PRT

<213> Artificial sequence

<400>1

His Lys Gln Leu Val Phe Phe Glu Glu Asp

1 5 5 10

<210>2

<211>10

<212>PRT

<213> Artificial sequence

<400>2

His Lys Gln Leu Pro Phe Phe Glu Glu Asp

1 5 10

<210>3

<211>10

<212>PRT

<213> Artificial sequence

<400>3

His Gln Lys Leu Val Phe Phe Ala Glu Asp

1 5 10

<210>4

<211>42

<212>PRT

<213> Human (Homo sapiens)

<400>4

Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys

1 5 10 15

Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile

20 25 30

Gly Leu Met Val Gly Gly Val Val Ile Ala

35 40

Claims (3)

1. A polypeptide consisting of the following amino acid sequence: His-Lys-Gln-Leu-Pro-Phe-Phe-Glu-Glu-Asp (SEQ ID NO: 2). 2. Use of the polypeptide according to claim 1 in the preparation of a medicament for preventing and/or treating Alzheimer's disease. 3. A pharmaceutical composition comprising the polypeptide according to claim 1 and a pharmaceutically acceptable carrier.

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